Sleeping beauty mutase (sbm) is expressed and interacts with ygfd in Escherichia coli.
In Escherichia coli, a four-gene operon, sbm-ygfD-ygfG-ygfH, has been shown to encode a putative cobalamin-dependent pathway with the ability to produce propionate from succinate in vitro [Haller T, Buckel T, Retey J, Gerlt JA. Discovering new enzymes and metabolic pathways: conversion of succinate to propionate by Escherichia coli. Biochemistry 2000;39:4622-4629]. However, the operon was thought to be silent in vivo, illustrated by the eponym describing its first gene, "sleeping beauty mutase" (methylmalonyl-CoA mutase, MCM). Of the four genes described, only ygfD could not be assigned a function. In this study, we have evaluated the functional integrity of YgfD and Sbm and show that, indeed, both proteins are expressed in E. coli and that YgfD has GTPase activity. We show that YgfD and Sbm can be co-immunoprecipitated from E. coli extracts using antibody to either protein, demonstrating in vivo interaction, a result confirmed using a strain deleted for ygfD. We show further that, in vitro, purified His-tagged YgfD and Sbm behave as a monomer and dimer, respectively, and that they form a multi-subunit complex that is dependent on pre-incubation of YgfD with non-hydrolysable GTP, an outcome that was not affected by the state of Sbm, as holo- or apoenzyme. These studies reinforce a role for the in vivo interaction of YgfD and Sbm.
Froese
D S
DS
Department of Biochemistry and Molecular Biology, University of Calgary, 3330 Hospital Drive NW, Calgary, AB, Canada T2N 4N1. sfroese@ucalgary.ca
Dobson
C M
CM
White
A P
AP
Wu
X
X
Padovani
D
D
Banerjee
R
R
Haller
T
T
Gerlt
J A
JA
Surette
M G
MG
Gravel
R A
RA
D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D008765:Methylmalonyl-CoA Mutase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009876:Operon; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid
D029968:Escherichia coli Proteins; D020558:GTP Phosphohydrolases; C539645:YfgD protein, E coli; D008765:Methylmalonyl-CoA Mutase